Next Article in Journal
Pathophysiology of Diet-Induced Acid Stress
Previous Article in Journal
Mesenchymal Transglutaminase 2 Activates Epithelial ADAM17: Link to G-Protein-Coupled Receptor 56 (ADGRG1) Signalling
Previous Article in Special Issue
Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 4
10.3390/ijms25042333
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain

by
Maitane Aranzamendi
1,2,
Kyriaki Xanthopoulou
3,4,
Sandra Sánchez-Urtaza
2,
Tessa Burgwinkel
3,4,
Rocío Arazo del Pino
3,4,
Kai Lucaßen
3,4,
M. Pérez-Vázquez
5,
Jesús Oteo-Iglesias
5,
Mercedes Sota
6,
Jose María Marimón
1,
Harald Seifert
3,4,7,
Paul G. Higgins
3,4,* and
Lucía Gallego
2,*
1
Respiratory Infection and Antimicrobial Resistance Group, Microbiology Department, Infectious Diseases Area, Biogipuzkoa Health Research Institute, Osakidetza Basque Health Service, Donostialdea Integrated Health Organization, 20014 San Sebastián, Spain
2
Acinetobacter baumannii Research Group, Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, University of the Basque Country UPV/EHU, 48940 Leioa, Spain
3
Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50935 Cologne, Germany
4
German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, 50935 Cologne, Germany
5
National Center of Microbiology, Reference and Research Laboratory for Antibiotic Resistance, ISCIII, Centro de Investigación Biomédica en Red, Enfermedades Infecciosas (CIBERINFEC), 28220 Madrid, Spain
6
Clinical Laboratory Management Department, IIS Biodonostia Health Research Institute, University Hospital Donostia, 20014 Donostia, Spain
7
Institute of Translational Research, CECAD Cluster of Excellence, University of Cologne, 50935, Cologne, Germany
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(4), 2333; https://doi.org/10.3390/ijms25042333
Submission received: 3 January 2024 / Revised: 9 February 2024 / Accepted: 13 February 2024 / Published: 16 February 2024
(This article belongs to the Special Issue Antimicrobial Resistance, Molecular Epidemiology and Emerging Therapeutic Strategies in Carbapenem-Resistant Gram-Negative Bacteria)
Browse Figures
Review Reports Versions Notes

Abstract

:
Infections caused by carbapenem-resistant Acinetobacter baumannii are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two blaOXA24/40 Acinetobacter baumannii outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3Pas/ST106Oxf (IC3) containing blaOXA24/40, blaOXA71 and blaADC119. Outbreak 2 isolates were exclusively ST2Pas/ST801Oxf (IC2) blaOXA24/40, blaOXA66 and blaADC30, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the blaOXA24/40 gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.

1. Introduction

Acinetobacter baumannii was listed in 2008 as part of the broad ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species) [1,2] and it has been recognized as such a big threat, that the World Health Organization (WHO) in 2017 classified carbapenem-resistant A. baumannii (CRAb) as one of the “Priority 1: Critical group” organisms for which new antibiotics are urgently needed [3,4].
A. baumannii is an opportunistic pathogen frequently responsible for outbreaks in health-care facilities, particularly in Intensive Care Units (ICUs) [5]. A wide range of virulence factors are responsible for pathogenesis and high mortality of A. baumannii, contributing to pathogen survival in stressful conditions [6,7,8,9]. A. baumannii can also easily acquire antibiotic resistance determinants rendering multiple antimicrobials useless, hindering patient treatment [10,11]. To add to this, the organism’s ability to survive under a wide range of environmental conditions and to persist for extended periods of time on dry surfaces make it a frequent endemic nosocomial pathogen [12,13,14,15].
Moreover, the persistence of A. baumannii in the hospital environment could potentially be favoured by reduced susceptibility to commonly used biocides and this seems to induce co-resistance to some antimicrobial agents (carbapenems, aminoglycosides, quinolones and tetracyclines). These two factors, persistence and multidrug resistance (MDR), have been clearly associated with the epidemic behaviour of some successful clones of A. baumannii [16].
High resistance rates to last resort antibiotics, such as carbapenems, are of major concern [17]. OXA enzymes are the most prevalent carbapenem-resistance determinant in A. baumannii with up to six families described i.e., the intrinsic OXA-51 and the acquired OXA-23, OXA-24/40, OXA-58, OXA-143 and OXA-235 [18]. The OXA-24/40 carbapenemase was first identified in a multidrug-resistant isolate from a hospital in Northern Spain [19] but quickly was confirmed as an endemic enzyme in the Iberian peninsula [20]. Although first described as chromosomally encoded, it is also harboured by plasmids [21].
International Clone 2 (IC2) is the most prevalent clone in most countries of Southern Europe and in the United States, whereas other less prevalent clones have been described in Central and South America (IC5 and IC7) and in Africa and the Middle East (IC9) [22,23,24,25,26]. In Spain IC2 isolates are the predominant genotype and have been associated with increased antimicrobial resistance (AMR), especially to carbapenems due to the presence of OXA-type carbapenemases, mainly OXA-23 and less frequently OXA-24/40 [27,28,29].
Although significant advances have been made in our understanding of A. baumannii over the years, many unanswered questions about the spread and evolution of carbapenem-resistant isolates remain unclear [30]. The aim of the present study was to investigate the molecular epidemiology and genetic features of two OXA-24/40-producing CRAb outbreaks at a large tertiary hospital in Northern Spain in 2009 and 2019–2021, respectively.

2. Results

2.1. Species Identification and Antimicrobial Susceptibility

All isolates were confirmed as A. baumannii according to the MALDI-TOF, gyrB multiplex PCR, and identification of the intrinsic blaOXA-51. Antimicrobial susceptibility testing showed values above the EUCAST breakpoints (Version 13.1, 2023) for all β-lactams (including imipenem and meropenem, MICs of >8 mg/L and of >4 mg/L respectively), aminoglycosides (MICs of >4 mg/L against gentamicin and tobramycin except for HUC-106 and HUC-109 with MICs of 4 mg/L; MICs of >8 mg/L against amikacin for the 86.8% of the isolates), fluoroquinolones (MICs of >1 mg/L against ciprofloxacin and levofloxacin), sulphonamides (MICs of >4/76 mg/L against cotrimoxazole except for HUC-106 and HUC-109 with MICs of 2/38 mg/L), while all the isolates except for HUC-88 were susceptible to colistin (MIC ≤ 2 mg/L) (Tables S2 and S3).

2.2. MLST/cgMLST Analysis

The isolates were assigned to the Pasteur sequence type (ST) 2 (n = 24), ST3 (n = 12) and ST49 (n = 2) and following the Oxford scheme isolates were clustered in ST801 (n = 23), ST350 (n = 1), ST106 (n = 12) and ST128 (n = 2).
Using cgMLST analysis the isolates clustered with previously identified international clones (IC). In detail the isolates recovered in 2009 were identified as IC3 (n = 11), IC2 (n = 2) and unique (n = 2). The IC3 isolates formed a cluster of closely related isolates differing in up to 8 alleles. Interestingly, the control isolate HUC-A11 (n = 1) recovered in 2002 clustered also together with the IC3 collected in 2009, suggesting that the latter isolates might have been involved in inter-hospital transmission.
Using cgMLST all isolates recovered between 2020–2021 and samples collected from potentially contaminated objects from 5 different patient rooms, belonged to IC2 and formed a cluster of closely related isolates differing in up to 2 alleles. It is worth mentioning that the two IC2 isolates collected in 2009 clustered together with the recovered between 2020–2021 and were considered as closely related. Concerning the control strains, we found that on one hand, although HUC-SM28 also belonged to IC2 it was 395 alleles different from the IC2 isolates from the outbreaks, so that, was considered unrelated. On the other hand, strain HUC-A11 isolated in 2002, which was also identified as IC3, clustered with IC3 isolates from Outbreak 1 (Figure 1, in cyan).

2.3. Resistome

Different variants of the blaOXA-51 gene were found; blaOXA-66 (IC2), blaOXA-71 (IC3) and blaOXA-98. All isolates encoded the carbapenemase blaOXA-24/40 gene, and a novel blaOXA-24/40-like variant, named blaOXA-1040 (GenBank Accession Number OK271078), identified for the first time in isolate HUC-A11 (Table 1). One isolate (HUC-101) also harboured a blaOXA-58 gene.
Two variants of intrinsic blaADC-51 (blaADC-30 and blaADC-119) were predomminantly detected in the isolates, with the variants reflecting the cgMLST cluster pattern, i.e., blaADC-30 was found in all IC2 isolates, while blaADC-119 was associated with IC3. Furthermore, the intrinsic blaADC-1 and blaADC-241 variants were associated with clonal lineages IC2 and NA respectively, as confirmed by Oxford and Pasteur MLST, as well as cgMLST.
Furthermore, the IC3 isolates encoded the aadB-like, aph(3′)-Via and sul1, while all the IC2 isolates encoded the aac(3)-Ia-like, aadA1, aph(3′)-VIa-like, strA and strB, the sulphonamide resistance gene sul2 in addition to sul1, as well as the tetracycline resistance determinant tet(B)-like. All fluoroquinolone resistant isolates showed a double substitution Ser81-Leu and Ser84-Leu in GyrA and ParC, respectively (Table 1).

2.4. Virulome

Results are summarised in Table 2. We found genes coding for the Acinetobacter trimeric autotransporter (ata); host cell adhesion (pil and fim); the two-component signal transduction system, biofilm controlling response regulator and sensor kinase (bfmRS); efflux pump operon (adeFGH); the quorum-sensing system (abaIR), consisting of the acyl-homoserine-lactone synthase AbaI and the DNA-binding HTH-domain-containing protein AbaR; the chaperonusher pilus CsuC/D/E (csuCDE); the biofilm-associated protein Bap (bap) as well as the Bap-like proteins Blp1 and Blp2 (bpl1, bpl2); the outer membrane protein A (ompA); the polysaccharide poly-β-(1,6)-N-acetyl glucosamine (PNAG) encoded by gene cluster pgaABCD; and the coagulation targeting metallo-endopeptidase of A. baumannii (cpaA).
To mention, the chaperonusher pilus CSU-encoding genes were only detected in IC2, while capsular gene cpaA was only present in isolates HUC-106 and HUC-109. Although most of them were homogeneously present or not in all the isolates of each clone, ata gene was detected in the 78% of IC2 isolates, bfmS in the 82% of IC3 isolates and bap gene was present in the 36% of isolates of IC3. The K32, K1 and K11 capsular types, and OCL5, OCL1 and OCL8 outer core loci were found in our IC2, IC3 and NA strains, respectively.
The most important virulence genes in A. baumannii related to adherence, biofilm formation, exotoxins, exoenzymes, iron uptake, capsular polysaccharide and lipooligosaccharide outer core loci are summarized in Table 2.

2.5. OXA-24/40 Encoding Plasmids

Using a hybrid assembly approach, it was identified that the blaOXA-24/40 was encoded on a plasmid of 8.9 Kb in size in the isolates HUC-90, HUC-99 and Aba-1527 (Figure 2). The OXA-24/40-encoding plasmid was identical in the isolates belonging to IC2 clone (HUC-99 and Aba-1527) with a 100% coverage and identity, and highly similar to the plasmid of the isolate belonging to IC3 (HUC-90) with a 100% coverage and 99% identity.
Plasmid annotation revealed ORFs encoding for the carbapenemase OXA-20/40; a RepM replicon (Rep-3 superfamily, GR2); a toxin-antitoxin system (BrnT-BrnA) that is involved in vertical stability; TonB-dependent receptor, related to the transmission of signals from the outside of the cell leading to transcriptional activation of target genes; DIP1984 family protein encoding a hypothetical protein as well as three unidentified hypothetical proteins.

2.6. Transfer of blaOXA-24/40 by Electroporation and Conjugation Experiments

The three blaOXA24/40-encoding plasmids (pHUC-90, pHUC-99 and pAba-1527) were transferable by electroporation into A. baumannii ATCC17978 and the presence of blaOXA24/40 gene in the obtained transformants was confirmed by PCR. Furthermore, conjugation experiments showed that the OXA-24/40-encoding plasmids present in the IC2 isolates, HUC-99 and HUC-86/Aba1527, were transferred into the recipient A. baumannii BM4547. Conjugation experiments with the control strains were not successful.

3. Discussion

The relevance of carbapenem-resistant A. baumannii as a healthcare associated pathogen (HAP) is in part due to its ability to survive for long periods in hospital environment; therefore, it should be taken into account as a relevant source of A. baumannii outbreaks [13,31].
The present study describes two independent outbreaks of isolates harbouring the blaOXA-24/40 gene caused by two different clones in 2009 and 2020, belonging to IC3 and IC2, respectively.
Isolates showed a multidrug resistance phenotype, and besides phenotypical resistance to β-lactams, including carbapenems, most of the isolates of our collection were also resistant to sulphonamides, aminoglycosides and fluroquinolones, restricting the therapeutic options to colistin (except for isolate HUC-88, which was resistant to it), minocycline and tigecycline. Unfortunately, at the time of the outbreaks, susceptibility testing to novel compounds was not available in our hospital. Especially worrying is that most of the isolates in this study were obtained from the ICU section of the hospital.
Clonal relatedness analysis showed that the majority of the isolates of outbreak 1 belonged to IC3, more precisely they were assigned to ST3 (Pasteur) and ST106 (Oxford). This clone was considered a major clone in the past, but nowadays it has been replaced by IC2 and it is reported sporadically in Spain, United States of America and South Africa [32]. In contrast, isolates from outbreak 2 predominantly belonged to IC2, being ST2 (Pasteur) the most prevalent lineage, which is the most reported ST among CRAB isolates in the Mediterranean countries [33].
Outbreak 2 was caused by isolates belonging to IC2 and were identical to the IC2 isolates from outbreak 1, showing the persistence and stability of this strain over an eleven-year period. The homology of these isolates with the control strains showed coincidence between IC3 isolates from outbreak 1 with HUC-A11, isolated in 2002, but a wide distance (more than 300 alleles different) between IC2 isolates and HUC-SM28 recovered in 1999. It should also be noted that HUC-SM28 was the first A. baumanii isolate in which the blaOXA-24/40 carbapenemase variant was described (AF509241.1) [19,34]. In this study, sequencing of the blaOXA-24/40 gene in HUC-A11 isolate allowed us to identify a new variant of this gene, the blaOXA-1040 (OK271078).
Nowadays the genotype IC2 is the most commonly isolated clone in Mediterranean countries, especially in the ICU where it also has a higher rate of carbapenem resistance compared to the other A. baumannii international clones [35]. In the present study, isolates belonging to IC2 not only persisted along time, but also posessed multiple resistance determinants such as blaOXA-24/40, as well as resistance against aminoglycosides (aac(3)-Ia-like, aadA1,aph(3′)-VIa-like, strA and strB), sulphonamides (sul2 in addition to sul1), and tetracycline (tet(B)-like). Differences between IC2 and IC3 isolates relied on the absence of tet(B)-like, sul2, straA and straB, aac(3)-Ia, and the presence of aadB-like in isolates belonging to IC3 as well as the intrinsic OXA and ADC.
The success of A. baumannii as a nosocomial pathogen is due to not only antibiotic resistance, but also to virulence factors such as the capsule [36]. Interestingly, IC2 strains were KL32 capsular type that has been described as one of the most virulent loci. Moreover, KL32 has been associated with a significant increase of cell survival when treated with specific phages, and shows another way to favour long persistence, which may explain, among other factors, the long survival of the IC2 isolates of the present study [37,38].
Our results showed that, IC2 isolates in contrast to the other genotypes, harboured csuC, csuD and csuE genes and exhibited a higher percentage of bap genes, both csu and bap, known to be required in the early steps of biofilm formation. They also lack the cpaA gene, related to encapsulation, which conversely may even negatively impact bacterial biofilm formation by increasing cell surface hydrophobicity and may sensitize bacterial cells to UV radiation [39,40]. Isolates belonging to IC2 genotype usually harbour more virulence genes associated with higher capacity to produce biofilms on abiotic surfaces used for medical supplies, e.g., urinary catheters or endotracheal tubes what could reinforce the long persistence of our isolates [6,41,42,43,44,45].
Plasmids represent an ideal vehicle for acquiring and transferring resistance genes in A. baumannii. The most complex and large ones can carry transposable elements such as integrons or resistance islands (RI), while small plasmids may even lack conjugative or replicating genes, that may reduce the fitness cost of plasmid replication, enabling plasmid stability over long periods of time [46,47,48,49]. Our results showed an identical plasmid harbouring blaOXA-24/40 in the IC2 strains, HUC-99 and Aba1527, that persisted intact for a decade and crossed also the IC boundaries as it was also detected with minor variations in the IC3 isolate obtained in 2009. The plasmid belonged to GR2 homology group. Small plasmids of similar size have often been described in different clonal lineages, such as IC1 [48]. Furthermore, these plasmids were able to be transferred into the recipient strains used in this study, showing their role as dissemination mechanism of the blaOXA-24/40 gene.
There is a wide array of possibilities for A. baumannii to disseminate among patients and the hospital setting, including equipment, hospital surfaces, direct transmission between patients and/or healthcare workers, among the most relevant ones [13]. From 2009 to 2020, no CRAb isolates were recovered in our hospital, probably due to the additional measurements that were implemented such as an active screening programme on hospital admission, surveillance cultures from both patients and abiotic surfaces in critical hospital units, etc. [50,51,52].
The coronavirus disease 2019 (COVID-19) pandemic posed an additional threat and placed pressure on the increase in AMR worldwide, overwhelming health systems and creating a multifactorial problem. Hospitalization, especially in ICUs, increases the chances of healthcare associated infections (HAIs), mainly associated with the rise in the use of invasive devices, such as mechanical ventilation and vascular catheters. Increased length of stay, human resource challenges, that limited the adherence and effectiveness of standard infection prevention practices also contributed to the increase in HAIs [53,54,55]. Under this situation many opportunistic pathogens, such as A. baumannii, were able to survive in the nosocomial environment and re-emerged leading to fatal infections [54,55,56,57] as it could have happened during the pandemic, contributing to Outbreak 2. Unfortunately, we cannot ascertain whether the disruption of any measure could contribute in a larger degree or not to the re-emergence and spread in our hospital [2].
The high prevalence of antibiotic resistance as well as the complexity of intertwined resistance mechanisms in A. baumannii clinical isolates, highlights the importance of antimicrobial stewardship and active surveillance in clinics, as it poses a major threat to human health [58].

4. Materials and Methods

4.1. Bacterial Isolates

The molecular epidemiology of thirty-six OXA-24/40-positive CRAb isolates was investigated (Table S1) through a retrospective descriptive study. Twenty-nine isolates were recovered from 29 patients hospitalized at the Hospital Universitario de Cruces (Barakaldo, Spain) in 2009 (n = 15) and 2019–2021 (n = 14). The first isolate was selected, while isolates from sterile sites were chosen over respiratory samples, whenever possible. Additionally, seven samples collected in 2020 from potentially contaminated surfaces and equipment in the ICU of the same hospital were also studied. The environmental samples were processed using moist gauze following a modified method described previously [59]. As control strains we included two OXA-24/40 CRAb isolates obtained in 1999 (HUC-SM28) and in 2002 (HUC-A11) from two patients attended at the same hospital.

4.2. Species Identification, and Antimicrobial Susceptibility Testing

Species identification was performed using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) (Maldi Biotyper®; Bruker Daltonik, Bremen, Germany) and confirmed as A. baumannii by the gyrB multiplex PCR and identification of the intrinsic blaOXA-51-like gene, as previously described [60].
Susceptibility testing was performed using the VITEK®2 system with card AST-N089 22237 (bioMérieux, Marcy-l’Étoile, France) or Negative Combo 38 Panel from MicroScan® WalkAway® 96 Plus System (both Beckman Coulter Inc.; Brea, CA, USA). Minimal inhibitory concentrations (MICs) were interpreted using the resistance breakpoints for Acinetobacter spp. from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (Version 13.1, 2023; http://www.eucast.org/clinical_breakpoints/, accessed on 29 June 2023). For tigecycline, the EUCAST PK-PD (Non-species related) breakpoint of 0.5 mg/L for Enterobacterales was used.

4.3. DNA Extraction, PCRs, Short-Read Sequencing and Assembly

DNA was extracted using the Dneasy UltraClean Microbial Kit and used for the OXA-multiplex PCR to detect the carbapenemase-encoding genes blaOXA-51-like, blaOXA−23-like, blaOXA−58-like, blaOXA−24/40-like, blaOXA−143-like, and blaOXA−235-like, as previously described [61,62]. Subsequently, sequencing libraries were prepared using NEB Next® Ultra™ II FS DNA Library Prep Kit for Illumina® (#E7805//#6177; New England Biolabs) for a 250 bp paired-end sequencing run on an Illumina® MiSeq sequencer and the obtained reads were assembled de novo using the Velvet assembler integrated in the Ridom SeqSphere+ v.7.0.4 (Ridom GmbH, Münster, Germany).

4.4. Molecular Epidemiology and Determination of Antibiotic Resistance and Virulence Genes

The isolates were investigated by applying a validated core genome Multi-Locus Sequence Typing (cgMLST) scheme, using the Ridom SeqSphere+ v. 8.0.1 software (Ridom GmbH) to generate a minimum spanning tree including 2390 target alleles [63].
Sequence types (STs) were assigned using the Oxford and the Pasteur 7-loci multi-locus sequence typing (MLST) schemes from the genome assemblies using the pubMLST website (https://pubmlst.org/organisms/acinetobacter-baumannii, accessed on 5 October 2021).
The software Resfinder 4.4.1 (http://genepi.food.dtu.dk/resfinder, accessed on 2 September 2021) and CARD (https://card.mcmaster.ca/, accessed on 3 September 2021) were used to identify acquired resistance genes and the beta-lactamase database (http://www.bldb.eu/, accessed on 30 August 2021) was used to identify the blaADC variants.
Adherence, biofilm and quorum sensing-related genes search was performed using the PubMLST website, only exact matches were taken into consideration [64]. The Virulence Factor Database (VFDB) and Kaptive were used to identify exotoxins, exoenzymes, iron uptake, capsular polysaccharide and lipooligosaccharide outer core loci [65,66].

4.5. MinION Long-Read Sequencing and Assembly

DNA extraction for long-read sequencing was performed using the MagAttract kit (Qiagen Cat. No. 67653) and the Wizard Genomic Purification kit (Promega Cat. No. A2920) for the DNA isolation according to the manufacturer’s instructions. Libraries were prepared using the 1D Ligation Sequencing Kit (SQK-LSK109) in combination with Native Barcoding Kit (EXP-NBD104; Oxford Nanopore Technologies, Oxford, UK) and were loaded onto a R9.4 flow cell (Oxford Nanopore Technologies). The run was performed on a MinION MK1b device. Collection of raw electronic signal data and live base-calling was performed using the MinKNOW 22.10.10 and the Guppy basecaller (Oxford Nanopore Technologies).
Plasmid content of isolates HUC-90, HUC-99 and HUC-Aba1527 was investigated in detail by hybrid assembly combining the MiSeq short-reads with MinION long-reads using Unicycler v0.5.0.

4.6. Plasmid Annotation and Visualization

Circular plasmids were visualized using SnapGene 5.1.4.1 (GSL Biotech, Chicago, IL, USA) and were manually annotated.

4.7. Electroporation Experiments

To determine the transferability of blaOXA24/40, plasmid DNA was isolated from HUC-90, HUC-99 and HUC-Aba1527 using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) and electroporated into the reference strain A. baumannii ATCC 17978. Selection of A. baumannii transformants was performed on Luria-Bertani agar (Oxoid, Wesel, Germany) supplemented with ticarcillin (150 mg/L). The presence of blaOXA24/40 in the obtained transformants was confirmed by PCR.

4.8. Conjugation Experiments

The OXA-24/40-encoding plasmids of four isolates, HUC-SM28 (IC2/1999), HUC-90 (IC3/2009), HUC-99 (IC2/2009) and HUC-Aba1527 (IC2/2020), were investigated by broth-mate conjugation experiments to determine the transferability of the blaOXA-24/40 using the sodium azide-resistant Escherichia coli J53 and the rifampicin-resistant A. baumannii BM4547 as recipient strains. Selection of E. coli J53 transconjugants was performed using sodium azide (200 mg/L), and selection for A. baumannii BM4547 was performed using rifampicin (100 mg/L) both combined with ticarcillin (100 mg/L). The transconjugants were tested by PCR for the blaOXA-24/40 gene and were confirmed by Sanger sequencing the rpoB gene.

5. Conclusions

Our results illustrate not only the persistence of the IC2 clone over a period of 11 years in the same hospital setting resulting in a monoclonal outbreak in 2020, but also the stability of the plasmid harboring the blaOXA-24/40 gene in these isolates. Isolates belonging to this clone showed a high degree of multidrug resistance and a variety of virulence factors, what is a threat for patients, and can cause fatal infections.
These findings highlight the difficulty in eradicating A. baumannii in hospital settings and the need to strengthen surveillance measures and show that the emergence of blaOXA-24/40 is of high concern. The presence of environmental isolates belonging to IC2 containing blaOXA-24/40 gene, emphasizes the need to control the reservoir of resistant isolates to prevent outbreaks.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25042333/s1. Supplementary Table S1. Characteristics of clinical isolates and control strains included in this study; Supplementary Table S2. Antibiotic susceptibility profile of the CRAb isolates belonging to Outbreak 1 and control strains; Supplementary Table S3. Antibiotic susceptibility profile of the CRAb isolates belonging to Outbreak 2.

Author Contributions

Conceptualization, M.A., L.G., K.X. and P.G.H.; methodology, M.A., K.X., S.S.-U., R.A.d.P.; formal analysis, M.A., L.G., K.X., K.L., S.S.-U., J.M.M. and P.G.H.; investigation, M.A., K.X., T.B., R.A.d.P., K.L., M.P.-V.; resources, M.A., L.G., M.S., J.M.M., J.O.-I., H.S. and P.G.H.; data curation, M.A, K.X., R.A.d.P., M.P.-V. and P.G.H.; writing—original draft preparation, M.A., L.G., K.X., P.G.H. and S.S.-U.; writing—review and editing, M.A., L.G., K.X., P.G.H. and S.S.-U.; supervision, L.G. and P.G.H.; project administration, M.A., M.S., J.M.M., L.G., H.S. and P.G.H.; funding acquisition, M.A., L.G., M.S., J.M.M., H.S. and P.G.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Microbiology Department, Infectious Diseases Area, Respiratory Infection and Antimicrobial Resistance Group, Biogipuzkoa Health Research Institute and the DEPARTMENT OF EDUCATION OF THE BASQUE GOVERNMENT (Research Groups of the Basque University System 2021), grant number Group IT1578-22, GIC21/18. SS-U is funded by the Basque Government’s Postdoctoral Fellowship (2023–2026).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequence of the blaOXA-1040 gene is available in GenBank under the accession number OK271078. All the genomes sequences are available under the accession numbers included into the BioProject ID PRJNA1060652 (Table S1).

Acknowledgments

The authors acknowledge support given by the CAREME network (Control of Antimicrobial Resistance in the Mediterranean Area: Global Solutions for a Global Threat) and the Clinical Microbiology Department from the University Hospital Cruces, Biobizkaia Health Research Institute for technical support and donations in kind such as funds and materials used for experiments.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Vázquez-López, R.; Solano-Gálvez, S.G.; Juárez Vignon-Whaley, J.J.; Abello Vaamonde, J.A.; Padró Alonzo, L.A.; Rivera Reséndiz, A.; Muleiro Álvarez, M.; Vega López, E.N.; Franyuti-Kelly, G.; Álvarez-Hernández, D.A.; et al. Acinetobacter baumannii resistance: A real challenge for clinicians. Antibiotics 2020, 9, 205. [Google Scholar] [CrossRef]
  2. De Oliveira, D.M.P.; Forde, B.M.; Kidd, T.J.; Harris, P.N.A.; Schembri, M.A.; Beatson, S.A.; Paterson, D.L.; Walker, M.J. Antimicrobial resistance in ESKAPE pathogens. Clin. Microbiol. Rev. 2020, 33, e00181-19. [Google Scholar] [CrossRef] [PubMed]
  3. Rice, L.B. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: No ESKAPE. J. Infect. Dis. 2008, 197, 1079–1081. [Google Scholar] [CrossRef] [PubMed]
  4. Centers for Disease Control and Prevention (U.S.). Antibiotic Resistance Threats in the United States, 2019; Centers for Disease Control and Prevention (U.S.): Atlanta, GA, USA, 2019. [Google Scholar]
  5. Weinberg, S.E.; Villedieu, A.; Bagdasarian, N.; Karah, N.; Teare, L.; Elamin, W.F. Control and management of multidrug resistant Acinetobacter baumannii: A review of the evidence and proposal of novel approaches. Infect. Prev. Pract. 2020, 2, 100077. [Google Scholar] [CrossRef] [PubMed]
  6. Eze, E.; Chenia, H.; El Zowalaty, M. Acinetobacter baumannii biofilms: Effects of physicochemical factors, virulence, antibiotic resistance determinants, gene regulation, and future antimicrobial treatments. IDR 2018, 11, 2277–2299. [Google Scholar] [CrossRef]
  7. Harding, C.M.; Hennon, S.W.; Feldman, M.F. Uncovering the mechanisms of Acinetobacter baumannii virulence. Nat. Rev. Microbiol. 2018, 16, 91–102. [Google Scholar] [CrossRef] [PubMed]
  8. Sycz, G.; Di Venanzio, G.; Distel, J.S.; Sartorio, M.G.; Le, N.-H.; Scott, N.E.; Beatty, W.L.; Feldman, M.F. Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages. PLoS Pathog. 2021, 17, e1009802. [Google Scholar] [CrossRef] [PubMed]
  9. Badmasti, F.; Siadat, S.D.; Bouzari, S.; Ajdary, S.; Shahcheraghi, F. Molecular detection of genes related to biofilm formation in multidrug-resistant Acinetobacter baumannii isolated from clinical settings. J. Med. Microbiol. 2015, 64, 559–564. [Google Scholar] [CrossRef] [PubMed]
  10. Borges Duarte, D.F.; Gonçalves Rodrigues, A. Acinetobacter baumannii: Insights towards a comprehensive approach for the prevention of outbreaks in health-care facilities. APMIS 2022, 130, 330–337. [Google Scholar] [CrossRef]
  11. Hamidian, M.; Nigro, S.J. Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii. Microb. Genom. 2019, 5, e000306. [Google Scholar] [CrossRef]
  12. Katzenberger, R.H.; Rösel, A.; Vonberg, R.-P. Bacterial survival on inanimate surfaces: A field study. BMC Res. Notes 2021, 14, 97. [Google Scholar] [CrossRef] [PubMed]
  13. Cobrado, L.; Silva-Dias, A.; Azevedo, M.M.; Rodrigues, A.G. High-touch surfaces: Microbial neighbours at hand. Eur. J. Clin. Microbiol. Infect. Dis. 2017, 36, 2053–2062. [Google Scholar] [CrossRef]
  14. Blehm, C.J.; Monteiro, M.S.G.; Bessa, M.C.; Leyser, M.; Dias, A.S.; Sumienski, J.; Gallo, S.W.; da Silva, A.B.; Barros, A.; Marco, R.; et al. Copper-coated hospital surfaces: Reduction of total bacterial loads and resistant Acinetobacter spp. AMB Express 2022, 12, 146. [Google Scholar] [CrossRef]
  15. Ryu, S.Y.; Baek, W.-K.; Kim, H.A. Association of biofilm production with colonization among clinical isolates of Acinetobacter baumannii. Korean J. Intern. Med. 2017, 32, 345–351. [Google Scholar] [CrossRef] [PubMed]
  16. Fernández-Cuenca, F.; Tomás, M.; Caballero-Moyano, F.-J.; Bou, G.; Martínez-Martínez, L.; Vila, J.; Pachón, J.; Cisneros, J.-M.; Rodríguez-Baño, J.; Pascual, Á.; et al. Reduced susceptibility to biocides in Acinetobacter baumannii: Association with resistance to antimicrobials, epidemiological behaviour, biological cost and effect on the expression of genes encoding porins and efflux pumps. J. Antimicrob. Chemother. 2015, 70, 3222–3229. [Google Scholar] [CrossRef]
  17. Kyriakidis, I.; Vasileiou, E.; Pana, Z.D.; Tragiannidis, A. Acinetobacter baumannii antibiotic resistance mechanisms. Pathogens 2021, 10, 373. [Google Scholar] [CrossRef]
  18. Gupta, N.; Angadi, K.; Jadhav, S. Molecular characterization of carbapenem-resistant Acinetobacter baumannii with special reference to carbapenemases: A systematic review. Infect. Drug Resist. 2022, 15, 7631–7650. [Google Scholar] [CrossRef] [PubMed]
  19. Lopez-Otsoa, F.; Gallego, L.; Towner, K.J.; Tysall, L.; Woodford, N.; Livermore, D.M. Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a hospital in Northern Spain. J. Clin. Microbiol. 2002, 40, 4741–4743. [Google Scholar] [CrossRef]
  20. Da Silva, G.J.; Quinteira, S.; Bértolo, E.; Sousa, J.C.; Gallego, L.; Duarte, A.; Peixe, L.; Portugese Resistance Study Group. Long-term dissemination of an OXA-40 carbapenemase-producing Acinetobacter baumannii clone in the Iberian Peninsula. J. Antimicrob. Chemother. 2004, 54, 255–258. [Google Scholar] [CrossRef]
  21. Rosales, I.; Sevillano, E.; Gallego, L. Genetic mapping of the region containing the blaOXA-40 gene in isolates of Acinetobacter baumannii. Int. J. Antimicrob. Agents 2012, 39, 453–454. [Google Scholar] [CrossRef]
  22. Castanheira, M.; Mendes, R.E.; Gales, A.C. Global epidemiology and mechanisms of resistance of Acinetobacter baumannii-calcoaceticus complex. Clin. Infect. Dis. 2023, 76, S166–S178. [Google Scholar] [CrossRef]
  23. Higgins, P.G.; Hagen, R.M.; Kreikemeyer, B.; Warnke, P.; Podbielski, A.; Frickmann, H.; Loderstädt, U. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates from Northern Africa and the Middle East. Antibiotics 2021, 10, 291. [Google Scholar] [CrossRef]
  24. Silva, L.; Grosso, F.; Rodrigues, C.; Ksiezarek, M.; Ramos, H.; Peixe, L. The success of particular Acinetobacter baumannii clones: Accumulating resistance and virulence inside a sugary shield. J. Antimicrob. Chemother. 2021, 76, 305–311. [Google Scholar] [CrossRef]
  25. Holt, K.; Kenyon, J.J.; Hamidian, M.; Schultz, M.B.; Pickard, D.J.; Dougan, G.; Hall, R. Five decades of genome evolution in the globally distributed, extensively antibiotic-resistant Acinetobacter baumannii global clone 1. Microb. Genom. 2016, 2, e000052. [Google Scholar] [CrossRef]
  26. Di Popolo, A.; Giannouli, M.; Triassi, M.; Brisse, S.; Zarrilli, R. Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme. Clin. Microbiol. Infect. 2011, 17, 197–201. [Google Scholar] [CrossRef]
  27. Villar, M.; Cano, M.E.; Gato, E.; Garnacho-Montero, J.; Miguel Cisneros, J.; Ruíz de Alegría, C.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pascual, A.; et al. Epidemiologic and clinical impact of Acinetobacter baumannii colonization and infection: A reappraisal. Medicine 2014, 93, 202–210. [Google Scholar] [CrossRef] [PubMed]
  28. López, M.; Rueda, A.; Florido, J.P.; Blasco, L.; Fernández-García, L.; Trastoy, R.; Fernández-Cuenca, F.; Martínez-Martínez, L.; Vila, J.; Pascual, A.; et al. Evolution of the quorum network and the mobilome (plasmids and bacteriophages) in clinical strains of Acinetobacter baumannii during a decade. Sci. Rep. 2018, 8, 2523. [Google Scholar] [CrossRef] [PubMed]
  29. Xanthopoulou, K.; Urrutikoetxea-Gutiérrez, M.; Vidal-Garcia, M.; Diaz de Tuesta del Arco, J.-L.; Sánchez-Urtaza, S.; Wille, J.; Seifert, H.; Higgins, P.G.; Gallego, L. First report of New Delhi Metallo-β-Lactamase-6 (NDM-6) in a clinical Acinetobacter baumannii isolate from Northern Spain. Front. Microbiol. 2020, 11, 589253. [Google Scholar] [CrossRef] [PubMed]
  30. Cosgaya, C.; Ratia, C.; Marí-Almirall, M.; Rubio, L.; Higgins, P.G.; Seifert, H.; Roca, I.; Vila, J. In vitro and in vivo virulence potential of the emergent species of the Acinetobacter baumannii (Ab) Group. Front. Microbiol. 2019, 10, 2429. [Google Scholar] [CrossRef] [PubMed]
  31. Umezawa, K.; Asai, S.; Ohshima, T.; Iwashita, H.; Ohashi, M.; Sasaki, M.; Kaneko, A.; Inokuchi, S.; Miyachi, H. Outbreak of drug-resistant Acinetobacter baumannii ST219 caused by oral care using tap water from contaminated hand hygiene sinks as a reservoir. Am. J. Infect. Control. 2015, 43, 1249–1251. [Google Scholar] [CrossRef] [PubMed]
  32. Shelenkov, A.; Akimkin, V.; Mikhaylova, Y. International clones of high risk of Acinetobacter baumannii: Definitions, history, properties and perspectives. Microorganisms 2023, 11, 2115. [Google Scholar] [CrossRef]
  33. Cherubini, S.; Perilli, M.; Segatore, B.; Fazii, P.; Parruti, G.; Frattari, A.; Amicosante, G.; Piccirilli, A. Whole-genome sequencing of ST2 A. baumannii causing bloodstream infections in COVID-19 patients. Antibiotics 2022, 11, 955. [Google Scholar] [CrossRef] [PubMed]
  34. Bou, G.; Oliver, A.; Martínez-Beltrán, J. OXA-24, a novel Class D β-Lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain. Antimicrob. Agents Chemother. 2000, 44, 1556–1561, Erratum in Antimicrob. Agents Chemother. 2006, 50, 2280. [Google Scholar] [CrossRef] [PubMed]
  35. Garnacho-Montero, J.; Gutiérrez-Pizarraya, A.; Díaz-Martín, A.; Cisneros-Herreros, J.M.; Cano, M.E.; Gato, E.; Ruiz de Alegría, C.; Fernández-Cuenca, F.; Vila, J.; Martínez-Martínez, L.; et al. Acinetobacter baumannii in critically ill patients: Molecular epidemiology, clinical features and predictors of mortality. Enfermedades Infecc. Microbiol. Clin. 2016, 34, 551–558. [Google Scholar] [CrossRef] [PubMed]
  36. Ayoub Moubareck, C.; Hammoudi Halat, D. Insights into Acinetobacter baumannii: A review of microbiological, virulence, and resistance traits in a threatening nosocomial pathogen. Antibiotics 2020, 9, 119. [Google Scholar] [CrossRef] [PubMed]
  37. Schultz, M.B.; Pham Thanh, D.; Tran Do Hoan, N.; Wick, R.R.; Ingle, D.J.; Hawkey, J.; Edwards, D.J.; Kenyon, J.J.; Phu Huong Lan, N.; Campbell, J.I.; et al. Repeated local emergence of carbapenem-resistant Acinetobacter baumannii in a single hospital ward. Microb. Genom. 2016, 2, e000050. [Google Scholar] [CrossRef]
  38. Neto, S.; Vieira, A.; Oliveira, H.; Espiña, B. Assessing Acinetobacter baumannii virulence and treatment with a bacteriophage using zebrafish embryos. FASEB J. 2023, 37, e23013. [Google Scholar] [CrossRef]
  39. Nunez, C.; Kostoulias, X.; Peleg, A.; Short, F.; Qu, Y. A comprehensive comparison of biofilm formation and capsule production for bacterial survival on hospital surfaces. Biofilm 2023, 5, 100105. [Google Scholar] [CrossRef]
  40. Akoolo, L.; Pires, S.; Kim, J.; Parker, D. The capsule of Acinetobacter baumannii protects against the innate immune response. J. Innate Immun. 2022, 14, 543–554. [Google Scholar] [CrossRef]
  41. Espinal, P.; Martí, S.; Vila, J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfaces. J. Hosp. Infect. 2012, 80, 56–60. [Google Scholar] [CrossRef]
  42. Giannouli, M.; Antunes, L.C.; Marchetti, V.; Triassi, M.; Visca, P.; Zarrilli, R. Virulence-related traits of epidemic Acinetobacter baumannii strains belonging to the international clonal lineages I-III and to the emerging genotypes ST25 and ST78. BMC Infect. Dis. 2013, 13, 282. [Google Scholar] [CrossRef]
  43. Longo, F.; Vuotto, C.; Donelli, G. Biofilm formation in Acinetobacter baumannii. New Microbiol. 2014, 37, 119–127. [Google Scholar]
  44. Tomaras, A.P.; Dorsey, C.W.; Edelmann, R.E.; Actis, L.A. Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: Involvement of a novel chaperone-usher pili assembly system. Microbiology 2003, 149, 3473–3484. [Google Scholar] [CrossRef]
  45. Lin, F.; Xu, Y.; Chang, Y.; Liu, C.; Jia, X.; Ling, B. Molecular characterization of reduced susceptibility to biocides in clinical isolates of Acinetobacter baumannii. Front. Microbiol. 2017, 8, 1836. [Google Scholar] [CrossRef]
  46. Bertini, A.; Poirel, L.; Mugnier, P.D.; Villa, L.; Nordmann, P.; Carattoli, A. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2010, 54, 4168–4177. [Google Scholar] [CrossRef] [PubMed]
  47. Cerezales, M.; Xanthopoulou, K.; Wille, J.; Krut, O.; Seifert, H.; Gallego, L.; Higgins, P.G. Mobile genetic elements harboring antibiotic resistance determinants in Acinetobacter baumannii isolates from Bolivia. Front. Microbiol. 2020, 11, 919. [Google Scholar] [CrossRef] [PubMed]
  48. Lean, S.S.; Yeo, C.C. Small, enigmatic plasmids of the nosocomial pathogen, Acinetobacter baumannii: Good, bad, who knows? Front. Microbiol. 2017, 8, 1547. [Google Scholar] [CrossRef] [PubMed]
  49. Rafei, R.; Koong, J.; Osman, M.; Al Atrouni, A.; Hamze, M.; Hamidian, M. Analysis of pCl107 a large plasmid carried by an ST25 Acinetobacter baumannii strain reveals a complex evolutionary history and links to multiple antibiotic resistance and metabolic pathways. FEMS Microb. 2022, 3, xtac027. [Google Scholar] [CrossRef] [PubMed]
  50. Delgado Naranjo, J.; Villate Navarro, J.I.; Sota Busselo, M.; Martínez Ruíz, A.; Hernández Hernández, J.M.; Torres Garmendia, M.P.; Urcelay López, M.I. Control of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a hospital of the Basque Country after the introduction of environmental cleaning led by the systematic sampling from environmental objects. Interdiscip. Perspect. Infect. Dis. 2013, 2013, 582831. [Google Scholar] [CrossRef] [PubMed]
  51. Tacconelli, E.; Cataldo, M.A.; Dancer, S.J.; De Angelis, G.; Falcone, M.; Frank, U.; Kahlmeter, G.; Pan, A.; Petrosillo, N.; Rodríguez-Baño, J.; et al. ESCMID guidelines for the management of the infection control measures to reduce transmission of multidrug-resistant gram-negative bacteria in hospitalized patients. Clin. Microbiol. Infect. 2014, 20, 1–55. [Google Scholar] [CrossRef] [PubMed]
  52. Karampatakis, T.; Tsergouli, K.; Iosifidis, E.; Antachopoulos, C.; Volakli, E.; Karyoti, A.; Sdougka, M.; Tsakris, A.; Roilides, E. Effects of an active surveillance program and enhanced infection control measures on carbapenem-resistant gram-negative bacterial carriage and infections in pediatric intensive care. Microb. Drug Resist. 2019, 25, 1347–1356. [Google Scholar] [CrossRef]
  53. Kiffer, C.R.V.; Rezende, T.F.T.; Costa-Nobre, D.T.; Marinonio, A.S.S.; Shiguenaga, L.H.; Kulek, D.N.O.; Arend, L.N.V.S.; Santos, I.C. de O.; Sued-Karam, B.R.; Rocha-de-Souza, C.M.; et al. A 7-year Brazilian national perspective on plasmid-mediated carbapenem resistance in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii complex and the impact of the coronavirus disease 2019 pandemic on their occurrence. Clin. Infect. Dis. 2023, 77, S29–S37. [Google Scholar] [CrossRef] [PubMed]
  54. Meschiari, M.; Onorato, L.; Bacca, E.; Orlando, G.; Menozzi, M.; Franceschini, E.; Bedini, A.; Cervo, A.; Santoro, A.; Sarti, M.; et al. Long-term impact of the COVID-19 pandemic on in-hospital antibiotic consumption and antibiotic resistance: A time series analysis (2015–2021). Antibiotics 2022, 11, 826. [Google Scholar] [CrossRef] [PubMed]
  55. Editorial. Antimicrobial resistance in the age of COVID-19. Nat Microbiol 2020, 5, 779. [Google Scholar] [CrossRef]
  56. Petazzoni, G.; Bellinzona, G.; Merla, C.; Corbella, M.; Monzillo, V.; Samuelsen, Ø.; Corander, J.; Sassera, D.; Gaiarsa, S.; Cambieri, P. The COVID-19 pandemic sparked off a large-scale outbreak of carbapenem-resistant Acinetobacter baumannii from the endemic strains at an Italian hospital. Microbiol. Spectr. 2023, 11, e04505-22. [Google Scholar] [CrossRef] [PubMed]
  57. Cantón, R.; Gijón, D.; Ruiz-Garbajosa, P. Antimicrobial resistance in ICUs: An update in the light of the COVID-19 pandemic. Curr. Opin. Crit. Care 2020, 26, 433–441. [Google Scholar] [CrossRef]
  58. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis. Lancet 2022, 399, 629–655. [Google Scholar] [CrossRef]
  59. Corbella, X.; Pujol, M.; Argerich, M.J.; Ayats, J.; Sendra, M.; Peña, C.; Ariza, J. Environmental sampling of Acinetobacter baumannii: Moistened swabs versus moistened sterile gauze pads. Infect. Control Hosp. Epidemiol. 1999, 20, 458–460. [Google Scholar] [CrossRef]
  60. Cerezales, M.; Biniossek, L.; Gerson, S.; Xanthopoulou, K.; Wille, J.; Wohlfarth, E.; Kaase, M.; Seifert, H.; Higgins, P.G. Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in gram-negative bacteria within Germany. J. Med. Microbiol. 2021, 70, 001310. [Google Scholar] [CrossRef]
  61. Woodford, N.; Ellington, M.; Coelho, J.; Turton, J.; Ward, M.; Brown, S.; Amyes, S.; Livermore, D. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 2006, 27, 351–353. [Google Scholar] [CrossRef] [PubMed]
  62. Higgins, P.G.; Pérez-Llarena, F.J.; Zander, E.; Fernández, A.; Bou, G.; Seifert, H. OXA-235, a novel Class D β-Lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2013, 57, 2121–2126. [Google Scholar] [CrossRef] [PubMed]
  63. Zarrilli, R.; Pournaras, S.; Giannouli, M.; Tsakris, A. Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages. Int. J. Antimicrob. Agents 2013, 41, 11–19. [Google Scholar] [CrossRef] [PubMed]
  64. Jolley, K.A.; Bray, J.E.; Maiden, M.C.J. Open-access bacterial population genomics: BIGSdb Software, the PubMLST.Org website and their applications. Wellcome Open Res. 2018, 3, 124. [Google Scholar] [CrossRef]
  65. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef]
  66. Wyres, K.L.; Cahill, S.M.; Holt, K.E.; Hall, R.M.; Kenyon, J.J. Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive. Microb. Genom. 2020, 6, e000339. [Google Scholar] [CrossRef]
Ijms 25 02333 g001
Figure 1. Minimum spanning tree of the CRAb isolates belonging to Outbreak 1 (2009), Outbreak 2 (2020/2021) and control strain HUC-A11 based on 2390 target alleles (A. baumannii cgMLST). Numbers between the nodes indicate the number of alleles different. Isolates are coloured based on their year of isolation. Asterisks refer to environmental isolates.
Figure 1. Minimum spanning tree of the CRAb isolates belonging to Outbreak 1 (2009), Outbreak 2 (2020/2021) and control strain HUC-A11 based on 2390 target alleles (A. baumannii cgMLST). Numbers between the nodes indicate the number of alleles different. Isolates are coloured based on their year of isolation. Asterisks refer to environmental isolates.
Ijms 25 02333 g001
Ijms 25 02333 g002
Figure 2. Comparison of the three OXA24/40-encoding plasmids pHUC-90 (red), pAba-1527 (blue), and pHUC-99 (green). The figure was generated using the software BRIG: blast ring image generator.
Figure 2. Comparison of the three OXA24/40-encoding plasmids pHUC-90 (red), pAba-1527 (blue), and pHUC-99 (green). The figure was generated using the software BRIG: blast ring image generator.
Ijms 25 02333 g002
Table 1. Antimicrobial resistance determinants, MLST type, and clonal lineage of the investigated A. baumannii isolates. NA: not assigned.
Table 1. Antimicrobial resistance determinants, MLST type, and clonal lineage of the investigated A. baumannii isolates. NA: not assigned.
IsolateCollection DateMLSTClonal LineageAntibiotic Resistance Determinants
SToxSTpasSulphonamideBeta-LactamAminoglycosideTetracyclineFluoroquinolones
GyrAParC
HUC-8618 July 20098012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40, blaOXA-58		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
HUC-8817 February 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-893 June 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9026 June 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9218 September 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9313 August 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9412 August 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-962 July 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9717 June 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9826 June 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-9925 June 20098012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
HUC-10024 August 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-10124 August 20091063IC3sul1blaADC-119, blaOXA-71, blaOXA-24/40aadB-like,aph(3′)-VIa - S81LS84L
HUC-1069 October 2009128 related49NA - blaADC-241, blaOXA-98, blaOXA-24/40aadB-like - S81LWT
HUC-10922 June 2009128 related49NA - blaADC-241, blaOXA-98, blaOXA-24/40aadB-like - S81LWT
HUC-A1125 February 20021063IC3sul1blaADC-119, blaOXA-71, blaOXA-1040aadB-like,aph(3′)-VIa - S81LS84L
HUC-SM2826 April 19993502IC2sul1blaADC-1, blaOXA-66, blaOXA-24/40aac(3)-IIa-like,aph(3′)-VIa,strA,strBtet(B)-likeS81LS84L
Hi8737 December 20198012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba151611 June 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba151711 June 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba151815 June 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba15192 June 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152015 June 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152114 July 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152213 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152313 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152412 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152527 September 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152620 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152724 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152817 July 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba152916 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba153016 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba153130 September 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba153224 August 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba153323 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba153423 October 20208012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Aba15524 January 20218012IC2sul1,sul2blaADC-30, blaOXA-66,
blaOXA-24/40		aac(3)-Ia-like,aadA1,aph(3′)-VIa-like,strA,strBtet(B)-likeS81LS84L
Table 2. Virulence factors identified in each International Clone. ATA: Acinetobacter trimeric autotransporter; Type V pili: host cell adhesion; BCRR: Biofilm controlling response regulator; AdeFGH: efflux operon; Csu: chaperone-usher type pilus; BAP: Biofilm associated protein; OMPs: Outer membrane proteins; PNAG: Poly-N-acetyl-D-glucosamine; CPA: Coagulation targeting metallo-endopeptidase of A. baumannii. NA: not assigned. * ata gene was detected in the 78% of isolates. ** bfmS gene was detected in the 82% of isolates. *** bap gene was detected in the 36% of isolates.
Table 2. Virulence factors identified in each International Clone. ATA: Acinetobacter trimeric autotransporter; Type V pili: host cell adhesion; BCRR: Biofilm controlling response regulator; AdeFGH: efflux operon; Csu: chaperone-usher type pilus; BAP: Biofilm associated protein; OMPs: Outer membrane proteins; PNAG: Poly-N-acetyl-D-glucosamine; CPA: Coagulation targeting metallo-endopeptidase of A. baumannii. NA: not assigned. * ata gene was detected in the 78% of isolates. ** bfmS gene was detected in the 82% of isolates. *** bap gene was detected in the 36% of isolates.
ICAdherence and Biofilm ProductionExotoxinsExoenzymesIron UptakeCapsular PolysacharideLipooligosacharide Outer Core
ATAType V PiliBCRRAdeFGHQuorum SensingCsu FimbriaeBAPOMPsPNAGPhospholipases C and DCPAAcinetobactin
2ata *pil and fim genesbfmR, bfmSadeF, adeG, adeHabaI, abaRcsuC, csuD, csuEbapompApgaA, pgaB, pgaC, pgaDplc1, plc2, plcD-barA, barB, bas, and bauA genesKL32OCL5
3atapil and fim genesbfmS **adeF, adeG, adeHabaI, abaR - bap ***ompApgaA, pgaB, pgaC, pgaDplc1, plc2, plcD-barA, barB, bas, and bauA genesKL1OCL1
NAatapil and fim genesbfmR, bfmSadeF, adeG, adeHabaI, abaR - - ompApgaA, pgaB, pgaC, pgaDplc1, plc2, plcDcpaAbarA, barB, bas, and bauA genesKL11OCL8
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Aranzamendi, M.; Xanthopoulou, K.; Sánchez-Urtaza, S.; Burgwinkel, T.; Arazo del Pino, R.; Lucaßen, K.; Pérez-Vázquez, M.; Oteo-Iglesias, J.; Sota, M.; Marimón, J.M.; et al. Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain. Int. J. Mol. Sci. 2024, 25, 2333. https://doi.org/10.3390/ijms25042333

AMA Style

Aranzamendi M, Xanthopoulou K, Sánchez-Urtaza S, Burgwinkel T, Arazo del Pino R, Lucaßen K, Pérez-Vázquez M, Oteo-Iglesias J, Sota M, Marimón JM, et al. Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain. International Journal of Molecular Sciences. 2024; 25(4):2333. https://doi.org/10.3390/ijms25042333

Chicago/Turabian Style

Aranzamendi, Maitane, Kyriaki Xanthopoulou, Sandra Sánchez-Urtaza, Tessa Burgwinkel, Rocío Arazo del Pino, Kai Lucaßen, M. Pérez-Vázquez, Jesús Oteo-Iglesias, Mercedes Sota, Jose María Marimón, and et al. 2024. "Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain" International Journal of Molecular Sciences 25, no. 4: 2333. https://doi.org/10.3390/ijms25042333

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop